RNA World May Have Started Spinning with Triplet-Based Synthesis

RNA world models tend to stall at a crucial point: the genetic replication of folded RNA structures. Since these structures include ribozymes, the very elements that carry out replication, RNA world models need some sort of kickstart, a plausible mechanism whereby folded RNAs may be replicated.

Such a mechanism, suggest scientists based at the Medical Research Council (MRC), needn’t be anything like the one-base-at-a-time syntheses observed in today’s lifeforms. No, a three-base-at-a-time synthesis seems just the thing.

To support their contention that a triplet approach could overcome a paradox that has long bedeviled the RNA World Hypothesis, the MRC scientists described how they developed a ribozyme that can replicate folded RNAs, including itself. The ribozyme’s details appeared May 15 in the journal eLife, in an article entitled “Ribozyme-Catalysed RNA Synthesis Using Triplet Building Blocks.”

“We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer,” wrote the article’s authors. “The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication.”

Previously, scientists had developed ribozymes that could replicate straight strands of RNA, but if the RNA was folded, it blocked the ribozyme from copying it. Since ribozymes themselves are folded RNAs, their own replication is blocked. This paradox, the scientists of the current study argue, could be resolved by their ribozyme, which they say is the first engineered ribozyme that can “synthesize its own catalytic subunit '+' and '–' strands in segments and assemble them into a new active ribozyme.”

Normally when copying RNA, an enzyme would add single bases (C, G, A, or U) one at a time, but the new ribozyme uses three bases joined together, as a “triplet” (such as GAU). These triplet building blocks enable the ribozyme to copy folded RNA, because the triplets bind to the RNA much more strongly and cause it to unravel—so the new ribozyme can copy its own folded RNA strands.

The MRC scientists say that the “primordial soup” could have contained a mixture of bases in many lengths—one, two, three, four, or more bases joined together—but they found that using strings of bases longer than a triplet made copying the RNA less accurate.

“We found a solution to the RNA replication paradox by rethinking how to approach the problem,” explained the MRC’s Philipp Holliger, Ph.D., a senior author of the eLife paper. “We stopped trying to mimic existing biology and designed a completely new synthetic strategy. It is exciting that our RNA can now synthesize itself.

“These triplets of bases seem to represent a sweet spot, where we get a nice opening up of the folded RNA structures, but accuracy is still high. Notably, although triplets are not used in present-day biology for replication, protein synthesis by the ribosome—an ancient RNA machine thought to be a relic of early RNA-based life—proceeds using a triplet code.

The experiments were conducted in ice at –7°C, because the researchers had previously discovered that freezing concentrates the RNA molecules in a liquid brine in tiny gaps between the ice crystals. This also is beneficial for the RNA enzymes, which are more stable and function better at cold temperatures.

“This is completely new synthetic biology, and there are many aspects of the system that we have not yet explored,” Dr. Holliger continued. “We hope in future it will also have some biotechnology applications, such as adding chemical modifications at specific positions to RNA polymers to study RNA epigenetics or augment the function of RNA.”

The current work represents only a first step in developing a well-turned RNA world model, Dr. Holliger admitted: “Our ribozyme still needs a lot of help from us to do replication. We provided a pure system, so the next step is to integrate this into the more complex substrate mixtures mimicking the primordial soup—this likely was a diverse chemical environment also containing a range of simple peptides and lipids that could have interacted with the RNA.”